Anticytomegalovirus monoclonal antibodies and processes for the in vitro diagnosis of infections by human cytomegaloviruses and a protein-kinase inducible by cytomegaloviruses and recognisable by aforesaid monoclonal antibodies

ABSTRACT

The invention relates to a process for in vitro diagnosis of an infection by human cytomegaloviruses. The process consists of contacting cells, possibly carrying the infection, with a monoclonal antibody reacting with a polypeptide of molecular weight 68,000, induced by human cytomegalovirus and which possesses a protein-kinase activity. The detection of the reaction is preferably carried out by immunofluorescence.

[0001] The invention relates to human anticytomegalovirus monoclonalantibodies (HCMV) and to a process for the in vitro diagnosis ofinfections induced by human cytomegaloviruses It relates moreparticularly to antibodies of this type which are capable ofsimultaneously detecting human cytomegaloviruses (CMV), human cellsinfected by CMV and a polypeptide, particularly a protein induced byHCMV and having protein-kinase activity.

[0002] The invention relates also to the aforesaid polypeptide havingthe aforesaid protein-kinase activity.

[0003] It is known that CMV is the cause of numerous clinicalinfections, ranging between benign infectious manifestations, andcongenital manifestations, for example, particularly severemalformations.

[0004] CMV has also been recognised as a cause of morbidity andmortality in persons who have undergone organ transplants, for example,of the kidney. The detection of CMV is a problem which poses itselftherefore particularly acutely. In addition, the recognition andisolation of vaccinating polypeptides or proteins, or which can berendered vaccinating with respect to CMV would open up seriouspossibilities of prevention of said complications against which at thepresent time, few means of treatment are available.

[0005] Various authors have already brought their attention to bear ondiagnosis techniques, which could be based on the use of monoclonalantibodies with respect to the human cytomegalovirus. The monoclonalantibodies obtained hitherto, did not yet contribute the expectedsolution for performing a diagnosis, which is both easy and reliable, ofinfection with cytomegamoviruses.

[0006] In fact, it is known that CMV (a member of the family of virusesof herpes) induces in the infected cells a considerable number ofpolypeptides, at various stages of the infectious process, and atvarious levels of the infected cells. L. N. PEREIRA and Coll.(“Infection and Immunity”), June 1982, p. 924-932., report that theyhave produced a large number of hybridomas secretors of monoclonalantibodies, capable of recognising in previously unfixed cells,polypeptides, or glycoproteins induced by the CMV in cultures of thesecells. The differences observed by PEREIRA and Coll. at the level of therelative behaviour of different antibodies with respect to the virusitself, infected cells and proteins, glycoproteins, or polypeptides,isolatable from extracts of these cells, has led them to formulate thehypothesis, that the conformation of the viral glycoproteins recognisedby certain of these antibodies at the membranal surface of the cellsinfected by the CMV's, could be different from the conformation of theenvelope of the virion. The utilization of certain of the antibodiesisolated to perform diagnosis operations of the above-indicated type,has been evoked by these authors. It follows however from the foregoingand from the experimental results provided, that the monoclonalantibodies judged the most interesting, were also capable ofprecipitating several polypeptides at the same time. The use of theseantibodies to recognise antigenic determinants, or specific epitopes,could not therefore be contemplated by reason of the non-discriminatingcharacter of the recognition.

[0007] L. C. GOLDSTEIN and Coll. (“Infections and Immunity”, October1982, p. 273-281) also described a certain number of monoclonalantibodies which can recognise certain polypeptides localised in thenuclei, or in cytoplasmic inclusions of cells infected by CMV. Thesemonoclonal antibodies have been selected by their capacity to react withcells infected by CMV, and previously fixed with absolute methanol anddried in air. It does not however follow from the article that thesemonoclonal antibodies were adapted to recognise still intact infectedcells.

[0008] Lastly, C. AMEDEI and Coll. (Ann. Virol. (Institut Pasteur),describe a battery of 24 monoclonal antibodies directed against humancytomegalovirus. The larger number of monoclonal antibodies obtained,arises apparently from the techniques which have been used to detectthem. In particular, detection has employed immunofluorescent techniquesof detection of antibodies retained on previously infected cells, whichhad been fixed with acetone and dried in air. The same monoclonalantibodies are revealed to be practically devoid of activity withrespect to cells obtained from identical cell lines, previously infectedby the same virus, when these infected cells had been fixed beforehandwith methanol. Perhaps there should be sought in this observation, thecause of the low number of hybridomas secreting monoclonal antibodiesselective against cells infected with HCMV, which had been isolated byL. C GOLDSTEIN and Coll.

[0009] Among these monoclonal antibodies, some have been found to showneutralising properties with respect to several strains of virus. Thesemonoclonal antibodies have moreover been the subject of a French PatentApplication No. 83 05384, filed Mar. 31, 1983. These particularmonoclonal antibodies have been adapted for the carrying out of in vitrodiagnosis tests of an infection with HCMV. However, the PatentApplication and indeed, moreover, the above-mentioned article, have notrecognised among the various hybridomas described, that which isestablished in the following to secrete monoclonal antibodies,particularly effective for diagnosis operations, and suitable to permitthe isolation of a polypeptide inducible by HCMV's and endowed withbiological properties permitting a particularly refined diagnosisoperation.

[0010] It is an object of the invention to provide a method of diagnosisemploying monoclonal antibodies resulting from a selection amongst allof those which have been described in the state of the art. The use ofthese selected antibodies in in vitro diagnosis tests, is both easy andaccurate. They recognise a protein induced by HCMV's in still intactinfected cells in immunofluorescence tests. These antibodies are capableof providing positive responses during almost the whole of theinfectious cycle. The diagnosis can be improved and confirmed in dosagetests of the enzymatic activity. The infected cells producepolypeptides, proteins of glycoproteins, which are induced by theHCMV's. These polypeptides are moreover bearers of an epitope, orcontinuous sequential antigenic determinant. By the expression “bearerof an epitope or antigenic determinant”, must be understood adeterminant whose recognition by the antibodies is not connected withthe particular conformation of this site on the natural polypeptides,proteins or glycoproteins of the virus, or induced by the latter in thecells that it infects.

[0011] The invention results from the discovery which the inventors havemade, that the hybridoma secreting monoclonal antibodies previouslydescribed, complies with all of these conditions. More particularly, theinvention relates to the use of monoclonal antibodies produced by thesecreting hybridomas concerned for the detection in vitro of asequential antigenic determinant borne by a protein, of which themolecular weight is in the vicinity of 68,000, isolatable withoutdegradation from extracts of cells infected by a CMV strain, thisprotein possessing in addition, protein-kinase properties.

[0012] In other words, the human anticytomegalovirus monoclonal antibodyemployed in the process according to the invention for the in vitrodiagnosis of infections induced by human cytomegaloviruses and for thedetection of a protein-kinase inducible by human cytomegaloviruses inhuman cells, and particularly in human cell lines, can be defined by thecombination of its capacities:

[0013] to give rise to reactions detectable by immunofluorescence, onculture cells of human origin, previously infected with HCMV and fixedwith acetone

[0014] to react with essentially a single viral polypeptide induced byHCMV's in cells of human origin previously infected by them, thispolypeptide having a molecular weight of the order of 68,000, the bearerof a continuous sequential epitope, this polypeptide appearing in thenuclei and then diffusing, at least in part, into the cytoplasm of thesaid infected cells, from which it can be isolated.

[0015] preferably, to be fixed on protein A.

[0016] The invention relates, still more particularly, within the scopeof the above said applications, to monoclonal antibodies which recognisea polypeptide responding to the following characteristics/

[0017] It appears precociously in the cell (it is detectable 3 to 5hours after the adsorbtion of the virus on the cell);

[0018] it is specific to human CMV

[0019] it is accumulated in the cells infected by CMV; it persists atleast 4 days in the cells from the moment of its appearance (and thisparticularly in human fibroblasts of the MRC5 lung type) ; it possessesall of the properties of the preferred polypeptides, whosecharacteristics are indicated below.

[0020] The preferred monoclonal antibodies are those which do not reactwith non-specific receptors of IgG, and more particularly still, thosewhich are characterised by their capacity to detect the above-saidpolypeptide in cells infected by any HCMV, whatever the origin thereof,for example, strains known under the name Ad-169, Towne & Davis. Apreferred monoclonal antibody is that which is produced by the hybridomadeposited in the National Collection of Micro-Organisms and Cultures(C.N.C.M), under No. I-289 on Mar. 27, 1984.

[0021] The above said monoclonal antibodies are obtained by a processemploying hybridomas secreting monoclonal antibodies neutralising theHCMV viruses previously formed between cells of myelomas and spleencells of an animal previously immunised against a HCMV, such as thestrain HCMV Ad-169, this process being more particularly characterisedin that;

[0022] the said neutralising antibodies are made to react with humancells in cultures previously infected with an HCMV, and previously fixedwith acetone, then dried, preferably in air;

[0023] a first selection of those of the clones which give rise tofixation reaction, detectable by immunofluorescence, is carried out onsaid human cells after fixation of the latter with acetone and

[0024] when appropriate, a second selection of those of the monoclonalantibodies retained at the end of the first selection is carried out,which antibodies react selectively with a viral protein currentlyinduced in the nuclei of said infected cells, and which diffuse theninto the cellular cytoplasm, from which it can, as the case may be, beseparated, this protein having a molecular weight of the order of68,000, this protein possessing in addition, a protein-kinase activityand

[0025] the antibody selected is recovered from the reaction productobtained.

[0026] The recovery of the monoclonal antibody can be carried out in anymanner known in itself, for example by prior complexation of thereaction product formed with a protein A, particularly Staphylococcusaureus, by dissociation of the complex form in a suitable ionic buffer.

[0027] The at least partial isolation of the polypeptide of molecularweight 68,000, having a protein-kinase activity, can involve a priorseparation of the proteins or polypeptides, having different molecularweights, also contained in the cytoplasmic extracts, which can beobtained from the infected cells.

[0028] As regards more particularly the human cells utilisable for theproduction of the above-said polypeptide, recognised by the monoclonalantibodies produced by the hybridomas concerned above, it is possible toresort to any type of cell capable of supporting the replication of theHCMV. Cells whose use is advantageous, are constituted by human lungfibroblasts of the MRC-5 type.

[0029] The invention relates also to the polypeptides inducible by theHCMV's in infected cells, having a molecular weight which can reach avalue of the order of 68,000, a protein-kinase activity and bearing asequential epitope, these polypeptides being obtainable from a humancell culture, particularly human lung fibroblasts, previously infectedwith a HCMV culture, particularly of the strain Ad-169. This polypeptidecan be isolated from the cytoplasm of the infected cells. However, theinvention also relates to any polypeptide of lower molecular weight,also containing the sequential epitope recognised by the above-saidmonoclonal antibodies. It will be obvious to the specialist that, fromthe moment that the monoclonal antibody according to the invention isavailable, it becomes possible to envisage the isolation from theabovesaid polypeptide, having the above said molecular weight of about68,000, of smaller peptide sequences containing the same antigenicdeterminant, by resorting to techniques known in themselves, of cuttingup the intact initial peptide by enzymes capable of cleaving it atspecific sites. By way of examples of such proteins, may be mentionedthe enzyme of Staphylococcus Aureus V8, alpha-chymotrypsin, (mousesubmaxillary gland protease, marketed by the BOEHRINGER Company),collagenase of Vibrio alqinoluticus chemovar iophagus, which recognisesspecifically the dipeptides Gly-Pro and Gly-Ala.

[0030] It then becomes possible, from the peptides fragmented incontrolled manner by means of such enzymes, to detect those whichcontain the antigenic sites by reaction with the monoclonal antibody andwhich, as the case may be, also preserve the above-said protein-kinaseactivities.

[0031] Additional characteristics of the preferred polypeptidesaccording to the invention are as follows;

[0032] they possess a protein-kinase activity of the casein-kinase IItype; in particular, they are adapted to transfer phosphorus from ATP tocasein, and also to other substrates, such as phosvitine,glycogen-synthetase, histones and alpha-phosphorylases.

[0033] the activity of protein-kinase is inhibited by quercitine;

[0034] this polypeptide is capable of being autophosphorylized in vitro;

[0035] it is thermosensitive (loss of activity at −70° C. and at 100°C.).

[0036] The invention relates also more particularly to an in vitroprocess of diagnosis employing the above-said monoclonal antibodies,this process including the contacting of said monoclonal antibodies withthe cells of which the infected character or not is sought, and thedetection, preferably by immunofluorescence on the treated cells,without destruction of the latter, of the above-said polypeptide.Prefered conditions in which the test may be employed will be indicatedbelow.

[0037] The employment of these particular monoclonal antibodies indiagnostic tests is particularly advantageous, in that it can be carriedout very rapidly, as soon as there has been infection. It has in factbeen seen that the induced protein appears from 3 to 5 hours afterinfection, and persists throughout the infectious cycle. This propertydistinguises this monoclonal antibody very particularly from othermonoclonal antibodies. It is particularly useful in diagnostic tests, inas much as it is true that in practice, the moment of the initiation ofinfection is generally unknown, especially when the tests are carriedout on cells obtained from tissue biopsies, or other cell samplings inpatients suspected of being carriers of an infection withcytomegaloviruses

[0038] The monoclonal antibodies employed within the scope of theinvention may also be the basis of what may be considered as apurification process of a protein or glycoprotein polypeptide, or again,of a fragment of the latter, containing a sequential antigenicdeterminant of HCMV and/or possessing similar protein-kinase activities,from extracts or from lysates of human cell cultures, previouslyinfected with HCMV. In a prefered form of this process, recourse is hadto monoclonal antibodies of the type of those which have been definedabove, fixed previously, for example with cyanogen bromide to a solidsupport, such as the agarose lattice with three dimensionalcross-linking, marketed under the trademark SEPHAROSE by the SwedishCompany; PHARMACIA AG.

[0039] The continuous sequential character of the antigenic determinantof the polypeptides according to the invention results from the factthat the polypeptide bearing it can always be isolated from thecytoplasmic extract of infected cells, even when the separationoperation is carried out in the presence of powerful detergents likesodium dodecylsulfate (SDS), sodium deoxycholate, or the detergentmarketed under the trademark TRITON X-100, with or withoutbetamercaptoethanol. The above-mentioned agents are in fact known fortheir capacity to “undo” or to “unfold” a protein. Any “conformational”antigenic determinant would then cease to be recognised by themonoclonal antibody and this especially on the hypothesis that the“conformational site” involving amino acid sequences, which happened tobe in the same neighbourhood in the natural protein was only aconsequence of the closeness of sequences not directly linked with oneanother due to the initial conformation of said protein. It results fromthe foregoing that the protein or polypeptide which carries saidantigenic site, is apparently also immunogenic, and consequently adaptedto induce the production in vivo of antibodies neutralising the virusitself.

[0040] If the antibodies according to the invention enable the detectionspecifically of the protein of molecular weight about 68,000 in a cellextract, as the case requires protected against enzymatic degradations,to the exclusion of any other polypeptide or protein, having distinctmolecular weights, the consequence thereof is that this polypeptide orprotein will itself be useable in turn to carry out the selection andisolation of monoclonal antibodies capable of being produced byhybridomas obtained by cell fusions, bringing into action initiallyother types of myelomas, on the one hand, and other HCMV's for theimmunisation of the spleen cells intended to be fused with said myelomacells on the other hand.

[0041] Additional characteristics of the invention will appear also inthe course of the description which follows of examples of theproduction of hybridomas and of the isolation of those among them whichare adapted to produce antibodies according to the invention.

[0042] The techniques which have been employed in the performance ofthese examples have been carried out according to the followingoperational modes:

[0043] 1) Preparation of the Hybridomas

[0044] Mice were immunised by intraperitoneal injection of a suspensionof infected and unfrozen human MRC-5 cells. The cells had been frozenfive days after their infection with a strain of HCMV Ad-169. A boosterinjection was administered to these mice 3 or 4 weeks later. The micewere sacrificed and the spleens recovered for their cell fusion 3 daysafter the booster injection. The spleen cells were fused with cells ofmyeloma SP²/OAg 14 (SCHULMANN et coll. strain, “Nature”, 1978, 276,269/270) and the hybridomas formed were selected in an RPMI mediumcontaining hypoxantine (5 mM) and azaserine (1 mM) according to thetechnique described by R. CRAINIC et Coll., “Develop. Biol. Standard.”,1982, 50, 229-234, with respect to the production of hybridomassecreting monoclonal antibodies against the virus of poliomyelitis.

[0045] 2) Selection of Positive Hybridomas

[0046] The supernatant liquors of the hybridoma cultures thus retained,were selected for their capacity to secrete specific antibodies againstHCMV by indirect immunofluorescence, by means of late antigenpreparations. A series of positive hybridomas was cloned by the methodof limited dilutions, and the supernantants of these clones were thentested in the same manner by indirect fluorescence, by the methoddescribed in the article above mentioned of C. AMADEI et Coll. Thenthose of the hybridomas which secreted monoclonal antibodies havingcharacteristics similar to those of the monoclonal antibodies F6b ofC.AMADEI et Coll,. were selected. It is one of these strains which hasbeen deposited at the C.N.C.M., under N° I-289.

[0047] 3) Labeling of the Monoclonal Antibodies

[0048] The clones (2×10⁶ cells) were labeled either with ³⁵S-methionine,or with ³H-methionine, also by the technique described by C AMADEI etColl.

[0049] 4) Detection of the Fixation on the Protein A

[0050] It was again carried out by the method described in the articleof AMADEI et Coll.

[0051] The tests mentioned under paragraphs 3) and 4) were moreparticularly intended for the recognition of the ability of themonoclonal antibodies of producing a reaction detectable byimmunofluorescence with human cells in cultures previously infected withHCMV, and in addition, of their capacity of being precipitated byprotein A.

[0052] In order to detect among these monoclonal antibodies those whichare in addition capable of only reacting with a single polypeptide ofmolecular weight of the order of 68,000, it is preferred to carry out aprior radioactive labeling or the like of previously infected humancells, from which the cellular extracts are subsequently recovered. Itis these cellular extracts which will then be made to react with theunlabeled monoclonal antibodies to be selected.

[0053] 5) Extraction of the Antigen

[0054] Cells, whether infected or not, were washed twice in situ, with asaline solution buffered with phosphate (PBS) containing calcium andmagnesium ions (complete PBS), then recovered by scraping in completePBS containing phenyl-methyl-sulfonyl fluoride 10⁻⁴ M (PMSF) anddiisopropylfluoro-phosphate 10⁻⁴ (DFP), the cells being finallysubjected to extraction in a solution having a high salt content andhigh pH containing 0.5% of the detergent marketed under the name NP-40,by the technique of MICHELSON et Coll., (1979) J. Virol. 32, 259-267. Insome cases, the nuclei were separated from the cytoplasma after swellingof the cells in a hypotonic buffer, the addition of NP-40 (finalconcentration 0.5%) and subjection of the cell suspension to the actionof a plunger piston to break the cells (by the technique of MICHELSON etColl.). The suspension was then centrifuged at 800 g for 5 minutes at40° C. The nuclei were then retained in the pellet formed and thesupernatant constituting the cytoplasmic fraction was then recovered.The nuclei were subjected to the sane extraction treatment as mentionedabove.

[0055] All the extracts were centrifuged at 15,000 g for 15-30 minutesat 4° C. and frozen at −70° C. until the time of use. For the tests ofmeasurement of phosphotransferase activity, there followed the immediateimmunoprecipitation of the desired antigen, under the conditionsindicated below. The antigen can be preserved in the state bound toStaphylococcus aureus.

[0056] 6) Immunoprecipitation

[0057] The precipitation was carried out by employing 5 microliters ofascites fluid per 200 micrograms of protein extracted. The antigen andthe monoclonal antibodies (F6b) were incubated with stirring for 1.5hour at ambient temperature. The protein A was added in the form of a10% suspension of a strain of S. aureus serotype 1 of Cowan, inactivatedby heat and fixed with formaldehyde (50 microliters/5 microliters ofascites fluid). The antigenic antibody complexes were washed three timesin NEIS medium (KESSLER S. W. (1975) J. Immunol. 115, 1617-1624), thenstored in this buffer in the form of a 10% suspension at 4° C. Thecomplex can also be dissociated in an electrophoresis buffer by heatingfor five minutes at 100° C. The immunoprecipitate was analysed onsodium-dodecyl-sulfate-polyacrylamide electrophoresis gel (SDS-PAGE10%). The resolution of the proteins was carried out by differentialmigration with a current of 30-35 mA. When appropriate, fluorography wascarried out by the method of BONNAR et Coll. (1974), Eur. J. Biochem.46, 83-88, with the modification consisting of the use of a single bathbased on dimethylsulfoxide (DMSO) for 15 minutes at 37° C. and a singlebath of DMSO +20% of 2,5-diphenyloxazole (PPO) for 30 minutes at 37° C.

[0058] 7) Radioactive Labeling of the Cells

[0059] Infected cells were selected one hour subsequent to infection anddeprived of methionine for 15 minutes. Absorption with the virus wasthen carried out in a medium containing 10 microcuries/milliliter of³⁵S-methionine. For the other specimens of infected cells, the virus wasabsorbed for 1 hour and a growth medium was added. All the cells weredeprived of methionine for 15 minutes before being labeled for 2 hoursbefore their sampling at the respective times of 3, 9, 24, 48, 72 and 96hours after infection. The uninfected cells were labeled in the samemanner. The labeling medium consisted of an Eagle minimum essentialmedium, devoid of methionine, containing 10 microcuries, milliliter of³⁵S-methionine (AMERSHAM, specific activity 1300 curies/mmole). Thelabeling of the phosphoproteins was carried out over 3 hours in amodified DULBECCO medium, devoid of phosphate, containing 100microcuries/milliliter of ³²p-orthophosphate.

[0060] 8) Demonstration of the Protein-kinase Activity

[0061] Except when otherwise specified, the test for the protein-kinaseactivity was carried out by using 50 microliters of enzyme, bound tobacteria in a medium (final volume of 100 microliters) containingmagnesium phosphate buffer 22 mM at pH 6.8 KPi, magnesium sulfate MgSO₄5 mM, EDTA 0.15 mM, ATP 0.1 mM, 3 microcuries of ³²p-ATP, and 1milligram per milliliter of casein. The casein was not present in theanalysis tests of immunoprecipitates in the SDS-PAGE gel. Theimmunoprecipitates were then washed as indicated above, re-suspended inthe reaction mixture, and incubated for 30 minutes at 30° C. Thereaction was stopped with 10 microliters of EDTA solution 0.3 M. Theimmunoprecipitates were separated by centrifugation at 5000 g over 4minutes at 4°0 C. The supernatants were precipitated with a 5%trichloracetic acid solution (TCA). The precipitates were dissolved in a1 M soda solution, then reprecipitated with TCA, washed by filtration onfilters marketed under the name WHATMAN G.F/C. After additional washingin ethanol, the filters were dried and the pulses counted in a PACKARDscintillator liquid. The immunoprecipitates intended for analysis on gelwere washed 3 times in KPi buffer, re-suspended in the buffer of theelectrophoresis and analysed in 10% SDS/PAGE gel under the aboveindicated conditions.

[0062] 9) Estimation of the Sedimentation Coefficient

[0063] The antigen labeled with ³⁵S-methionine was prepared andextracted as indicated above from infected cells for 120 hours. Theantigen was then deposited at the upper part of a 5-25% sucrose gradientformed in PBS and subjected to centrifugation in a BECKMAN SW-41 rotorat 35,000 revolutions per minute for 17.25 hours at 4° C. Fractions werecollected. Each fraction was immunoprecipitated with F6b monoclonalantibody then analysed on SDS-PAGE.

[0064] 10) Electrophoresis of the Amino Acids Labeled with ³²p

[0065] After the protein-kinase reaction employing precipitates obtainedin the absence of casein, the product labeled with ³²p is detached witha 0.1% NH₄ HCO₃ solution containing 0.1% of SDS. The sample was driedunder vacuum, re-suspended in 6M HCl and hydrolysed overnight at 110° C.After evaporation of HCl, the preparations were re-suspended in theelectrophoresis buffer. The phosphoaminoacids were separated at pH 3.5,(in a medium formed from 50 milliliters of acetic acid and 5 millilitersof pyridine per liter) over 45 minutes under 1000V on thincellulose-coated plates (marketed by MACHEREY-NAGEL & Co). Standardswere dyed with the cadmium-ninhydrin reagent (described by DRICKAMER etColl., J. Biol. Chem. (1982) 257, 15156-1-161) and the plates wereexposed to a Kodak X-Omat film for one week.

[0066] The polypeptide immunoprecipitated by the monoclonal antibody F6bhad a molecular weight of about 68,000 (p68). It was immunoprecipitablefrom nuclei of infected cells from the third hour after infection. Itcan be immunoprecipitated also from cytoplasmic extracts 24 hours afterinfection. The content of this polypeptide with respect to the whole ofthe extracted proteins, 96 hours after infection, reached 0.5%(evaluation on the basis of the percentage of radioactivity contained inthe immunoprecipitate recovered). This polypeptide has a sedimentationcoefficient in water at 20° of 6.9 (by comparison with labels consistingof catalase and of bovine albumin serum).

[0067] The molecular weight of p63 was evaluated with respect to themigration distances in the same electrophoretic system, of the followingfour substances of known molecular weights

[0068] phospharylase A (94,000);

[0069] bovine serumalbumine (68,000);

[0070] fumarase (49,000)

[0071] aldolase (40,000).

[0072] The results witness the in vitro phosphorylation capacities ofthe polypeptide, which have been indicated above. The protein-kinasereaction with casein used as an acceptor, is done optimally at pH 6-6.5.It declines rapidly in media at more acid or more alkaline pH's. Thereaction depends on the presence of Mg²⁺ ions. These reactions are notcAMP-dependant. The phosphate transfer reaction is not operative in thepresence of manganese at high pH. This polypeptide (or this enzyme) isstable. It can be preserved in the form of a conjugate with S. aureus,without loss of activity after storage for 6 weeks at 4° C. Thecomplexation with F6b monoclonal antibodies does not result in loss ofactivity.

[0073] This enzyme can be autophosphorylated, the latter taking part atthe level of aminoacyl residues constituted by threonine and serine.

[0074] Polypeptides having molecular weights of the order of 68,000 andshowing the same characterisitics as those which have been indicatedabove, are induced in infected human cells, particularly lungfibroblasts, by other strains of cytomegalovirus, particularly humanstrains TOWNE & DAVIS. On the other hand cytomegalovirus strainsspecific to the monkey (COLBURN strain and SGC strain) do not induce theproduction of a protein of molecular weight 68,000 recognisable bymonoclonal antibodies secreted by the strain F6b.

[0075] The invention therefore relates more particularly to theselection of strains producing monoclonal antibodies having thecharacteristics of the F6b monoclonal antibodies.

[0076] The employment of this monoclonal antibody enables indeedefficient diagnosis in vitro of a viral infection by HCMV. It hasalready been indicated that this detection could be done practically atany moment of the infectious cycle, particularly by immunofluorescencereaction. This monoclonal antibody has also the great advantage that thediagnosis carried out by immunofluorescence can be confirmed, by thecapacity of the same antibody to form complexes with a protein inducedby human cytomegaloviruses which protein can then be detected owing toits protein-kinase activity The latter can be detected with a high levelof accuracy. Finally, the use of monoclonal antibodies of theabove-indicated type can be practiced in quantitative dosage tests ofthe level of infection of the human cells afflicted.

[0077] The invention relates also to the polypeptide itself, both as abiological reagent of great value, for example, in virus identificationtests, when their classification within cytomegaloviruses does not apriori seem certain. On the contrary, it can be used for the selectionof the most effective monoclonal antibodies for the constitution ofdiagnosis “kits”.

[0078] The invention relates also to “fits” for Performing in vitrodiagnoses as described above. Particularly a preferred “kit” comprises:

[0079] at least one microplate,

[0080] a preparation containing monoclonal antibodies suitable for thediagnosis,

[0081] control cellular extracts (obtained from human cells,particularly of the human MRC-5 type, these cells having or not beenpreviously infected with HCMV).

[0082] labeled antibodies (radioactive, enzymatic, fluorescent or otherlabel ) directed against immunoglobulins of human blood,

[0083] suitable buffers for the performance of

[0084] the various operations and for the final examination of theproducts which have reacted, to the fluorescence microscope.

[0085] Such an outfit will, for example, be employed in the followingmanner:

[0086] a cellular extract coming from a cellular biopsy to be examinedis deposited in one or several cups of the titration plate and adsorbedat their surface;

[0087] into the cups or wells of the microplate are introduced doses ofthe monoclonal antibody preparation;

[0088] the plate is placed to incubate at a suitable temperature;

[0089] the microplate is then efficiently washed;

[0090] then there is introduced into the cups of the microplate labeledantibodies directed against blood immunoglobulins, hence against themonoclonal antibodies, and it is again incubated;

[0091] the label is detected (for example by action on the suitablesubstrate when the label is constituted by an enzyme),

[0092] all of these operations being also carried out with respect tocontrol cell extracts, and this for the purposes of comparison.

[0093] Advantageously, it is possible also to carry out detection testsand quantitative dosage tests, for example, by passage of a cellularextract obtained under conditions for example of the type of those whichhave been envisaged above, on an affinity chromatography columncomprising the monoclonal antibodies fixed to agarose gell, for example,as has been indicated above, the dissociation of the complex possiblyformed between the monoclonal antibodies used, and the polypeptidespecifically recognised by the latter, and the quantitative dosage ofthe polypeptide, by employing its protein-kinase properties.

[0094] As is self-evident and as emerges already from the foregoing, theinvention is in no way limited to those of its types of application andembodiments which have been more especially envisaged; it encompasses onthe contrary all modifications.

1. A process for the detection of an infection, particularly for in vitro diagnosis, by human cytomegalovirus in cells of human origin, which comprises contacting either intact cells or extracts obtained from these cells, with a monoclonal antibody defined by the combination of the following properties: it gives rise to reactions detectable by aminofluorescence, on culture cells of human origin, previously infected with HCMV and fixed with acetone; it reacts essentially with a single viral polypeptide induced by HCMV's in cells of human origin previously infected by them, this polypeptide having a molecular weight of the order of 68,000 and bearing a continuous sequential epitope, said polypeptide having the further properties of appearing in the nuclei and of then diffusing at least in part from said nuclei into the cytoplasm of said infected cells.
 2. The process of claim 1 which comprises contacting said cells or cell extracts with a monoclonal antibody which is further capable of reacting with protein A.
 3. The process of claim 1 wherein said monoclonal antibody is one which reacts with a polypeptide which has a molecular weight of about 68,000; which possesses a protein-kinase II activity; which appears from 3 to 5 hours after adsorption of the virus on the cells and which, as from the time of its appearence accumulates and persists at least over 4 days in the infected cells.
 4. The process of claim 3 which is carried out on an extract of said cells and which comprises contacting said extract with said monoclonal antibody to form a complex between said monoclonal antibody and said polypeptide, recovering said polypeptide from said complex and measuring the protein-kinase activity of said polypeptide.
 5. Polypeptide inducible in cells of human origin by a human cytomegalovirus and having the following characteristics: it possesses a molecular weight of about 68,000; it possesses a protein-kinase activity of the casein-kinase II type; it is capable of causing transfer of phosphorus from ATP to casein, phosvitine, glycogen-synthetase, histones and alpha-phosphorylases; its protein-kinase activity is inhibited by quercitine; it is capable of being autophosphorylated in vitro; it is thermosensitive (loss of activity at −70° C. and at 100° C.
 6. The polypeptide of claim 5 which can form a complex with the monoclonal antibody of claim 1 .
 7. The polypeptide of claim 6 which can form a complex with the monoclonal antibody deposited at the C.N.C.M. under N° 1-289.
 8. The polypeptide of claim 5 , whose proteinkinase activity is dependent on the presence of Mg²⁺ ions, but which is dependent neither on the presence of manganese ions, nor on the presence of cAMP.
 9. The polypeptide of claim 5 , which is conjugated to protein A of Staphylococcus aureus.
 10. The polypeptide of claim 6 which is in the state of a complex with said monoclonal antibody.
 11. A kit for diagnosis of a cellular infection by human cytomegalovirus which comprises: a preparation containing monoclonal antibodies suitable for said diagnosis control cell extracts free of infection by HCMV, labelled antibodies directed against immunoglobulins of human blood and also capable of recognizing said monoclonal antibodies buffers suitable for enabling a complex between HCMV infected cells or an extract of said infected cells and said monoclonal antibody to be formed and detecting said complex when formed. 